Genetic mutations include genetically inherited germline mutation and somatic mutation that is acquired mutation induced in each cell, and it is reported that a specific genotype of a single nucleotide polymorphisms (SNP) of a specific gene in the germline mutation and somatic mutation such as point mutation (single base substitution), insertion, and deletion are associated with various diseases, and in recent years, identification of base sequences thereof is used for screening patients for which a specific drug is expected to be effective. For example, genetic polymorphism of UGT1A1 is used for judging a risk of occurrence of serious side effects of irinotecan, an anticancer agent. In a UGT1A1 genetic polymorphism test, it must be determined whether each of two base sequences (*6, *28) is the wild type without mutation, a heterozygote having both the wild type and the mutated forms, or a homozygote having only the mutated form. A JAK2 gene mutation used for diagnosing polycythemia vera, one of genetic mutations of myeloid proliferative diseases, is a gain-of-function acquired somatic mutation and is a point mutation of 1849 G>T of exon 14, resulting in constitutive activation of receptor tyrosine kinase. Since the detection of existence as well as quantitative changes of this point mutation has clinical utility, it is required to calculate an allele frequency. Therefore, as in genetic polymorphism detection, both the mutated form and the wild type must quantitatively be detected in point mutation detection. Additionally, the MPL (myeloproliferative leukemia virus) gene mutation set as World Health Organization (WHO) diagnostic criteria for primary myelofibrosis includes point mutations and deletion/insertion mutations at the 1543rd to 1544th bases of codon 515 of exon 10 and therefore has several mutation patterns at the same positions, and these patterns are desirably detected in a distinguished manner.
Ion-exchange chromatography is used as a method capable of accurately separating and detecting nucleic acid in a short time. An advantage of applying ion-exchange chromatography to detection of nucleic acid is that since nucleic acid can be separated according to the chain length thereof, multiple amplification products can be separated and detected in a single measurement by adjusting the length of the amplification products resulting from PCR (polymerase chain reaction), for example. Although this principle can theoretically be applied to the detection of multiple gene mutations as described above, ingenuity is required for detection of a slight difference of one base such as a single base substitution or a point mutation. In the case of single base substitution detection, even if primers for PCR are simply designed to bracket SNP sites to obtain amplification products, it is difficult to separate the difference of a single base by ion-exchange chromatography. In this regard, Patent Document 1 discloses a method for separating and detecting SNP with ion-exchange chromatography by adding to the 5′ end of an allele specific primer (ASP) a sequence (tag sequence) incompletely complementary to the template DNA, and thereby, artificially changing the length of amplification products resulting from PCR. However, if the added base sequence is too long, Tm value for primers significantly changes, so that specificity may no longer be maintained. Conversely, if the sequence is too short, a reduced difference in amplification product length leads to poor separation by ion-exchange chromatography, and it is concerned that single nucleotide polymorphism cannot accurately be determined.
On the other hand, it is reported that separation using capillary electrophoresis can be achieved by designing ASPs at the forward and the reverse sides on the double strand and by designing primers paired therewith in appropriate places other than the mutation site to obtain two kinds of amplification products having different sizes (Non-Patent Document 1). However, in this method, the primers irrelevant to mutation are paired with each other and allow amplification to proceed, so that components required for amplification are consumed, which may affect a specific reaction. Furthermore, since two pairs of paired primers are used, the efficiency of hybridization and amplification is prone to vary, which makes it difficult to accurately calculate an allele frequency when a gene mutation such as JAK2 gene mutation is detected. Additionally, this method is limited to detection of two kinds of mutations and cannot be applied to a large variety of mutations such as multiple mutations around codon 515 of MPL and point mutations of codon 12 and codon 13 of KRAS, NRAS, etc.